title: Characterization of Ceriporiopsis subvermispora bicupin oxalate oxidase expressed in Pichia pastoris.
abstract: Oxalate oxidase (E.C. 1.2.3.4) catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. Although there is currently no structural information available for oxalate oxidase from Ceriporiopsis subvermispora (CsOxOx), sequence data and homology modeling indicate that it is the first manganese-containing bicupin enzyme identified that catalyzes this reaction. Interestingly, CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC). We show that CsOxOx activity directly correlates with Mn content and other metals do not appear to be able to support catalysis. EPR spectra indicate that the Mn is present as Mn(II), and are consistent with the coordination environment expected from homology modeling with known X-ray crystal structures of OxDC from Bacillus subtilis. EPR spin-trapping experiments support the existence of an oxalate-derived radical species formed during turnover. Acetate and a number of other small molecule carboxylic acids are competitive inhibitors for oxalate in the CsOxOx catalyzed reaction. The pH dependence of this reaction suggests that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated.
authors.affiliation: Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, FL 32611-7200, USA.
authors.forename: Patricia
authors.initials: P
authors.lastname: Moussatche
authors.forename: Alexander
authors.initials: A
authors.lastname: Angerhofer
authors.forename: Witcha
authors.initials: W
authors.lastname: Imaram
authors.forename: Eric
authors.initials: E
authors.lastname: Hoffer
authors.forename: Kelsey
authors.initials: K
authors.lastname: Uberto
authors.forename: Christopher
authors.initials: C
authors.lastname: Brooks
authors.forename: Crystal
authors.initials: C
authors.lastname: Bruce
authors.forename: Daniel
authors.initials: D
authors.lastname: Sledge
authors.forename: Nigel G J
authors.initials: NG
authors.lastname: Richards
authors.forename: Ellen W
authors.initials: EW
authors.lastname: Moomaw
mesh_terms: D000085:Acetates / Q000378:metabolism; D002264:Carboxylic Acids / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D015870:Gene Expression; D008345:Manganese / Q000378:metabolism; D010070:Oxalates / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010843:Pichia / Q000235:genetics; D020072:Polyporales / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D000085:Acetates; D002264:Carboxylic Acids; D010070:Oxalates; D008345:Manganese; D010088:Oxidoreductases; C020598:oxalate oxidase